The long-term objective of this project is to understand and characterize the mechanism of mitochondrial DNA replication in Brassica campestris, a higher plant. Although much is known about the mechanism of DNA replication and the enzymes involved in replication of the compact animal mitochondrial genome (about 17 kbp), very little is known about mitochondrial DNA replication in other eukaryotic organisms. Yeast and other fungal mitochondrial genomes are up to about 5 times larger than the animal mitochondrial genome, while the mitochondrial genome in plants is even larger, ranging from 200-2500 kbp. The plant mitochondrial genome is capable of intramolecular recombination which generates subgenomic circles. It is not clear whether these subgenomic circles replicate autonomously, or whether only the unrecombined master mitochondrial genome replicates, followed by recombination to form the subgenomic circles. In order to address these points, B. campestris has been chosen because it has the smallest well-characterized mitochondrial genome in higher plants, and forms only two subgenomic circles of well- defined size. Complete physical and restriction maps are available, along with a complete clone bank. The specific methodologies to be used include electron microscopy analysis of total mitochondrial DNA from B. campestris to characterize the number and presence of replication intermediates on the three different sized molecules, mapping of origin regions by electron microscopy of restricted DNA, development of an in vitro DNA replication system to test specific B. campestris clones, and analysis of in vivo and in vitro replication intermediates by two-dimensional agarose gel electrophoresis. These efforts will provide a foundation for further studies to identify DNA sequences and proteins involved in mitochondrial DNA replication in B. campestris, and for understanding mitochondrial DNA replication in other eukaryotic systems which have a more complex genome than in animal cells.